A neuron is a main element controlling the life activity of an individual higher organism. It had been thought that neurons of the central nerve system perform neither postnatal differentiation nor regeneration, but only deciduate from one minute to the next. However in the 1990s, neuronal stem cells which had not yet differentiated into neurons were found in a fetal brain, and further the presence of neuronal stem cells in an adult brain was also proved. Thus, the possibility of regeneration of the central nerve system has been suggested. Now, possible therapy using neuronal stem cells for intractable neuronal diseases, and therapy using embryonic stem cells (ES cells, also called as universal cells which can differentiate into any type of cells) are stepping into the limelight.
However, since human neuronal stem cells can only be collected from human fetal brain resulting from artificial termination of pregnancy, there are ethical issues to surmount. Further, securing the dose required to treat neurological diseases is difficult because the amount of such stem cells that can be collected is limited. Furthermore, there is another problem that only a portion of neuronal stem cells collected with considerable effort, differentiate into neurons and most of them differentiate into glial cells.
Regarding these problems, we have considered that VHL gene and VHL gene products may play a role from the developmental stage of neurons based on the fact that they are specifically expressed in neurons of the central nerve system. Accordingly, our studies on expression of VHL gene products in neuronal stem cells have revealed that VHL gene products are mainly expressed in the cytoplasm as neuronal stem cells differentiate into neurons. Further, introduction of VHL gene into neuronal stem cells using a herpes simplex virus vector resulted in promotion of differentiation into neurons. Hence, we have shown that VHL gene is a gene involved in induction of neuronal differentiation in neuronal stem cells (Kanno H: Cancer Res 2820-4, 2000). However, neuronal stem cells are known from the beginning to differentiate into either neurons or glial cells. Addition of basic fibroblast growth factor (bFGF) into a medium also results in a similar phenomenon. Therefore, whether or not VHL gene itself possesses an ability to induce direct differentiation into nerves remains unknown.
On the other hand, unlike neuronal stem cells, immortalized cancer cells or ES cells can be cultured in vitro infinitely. However, the mechanism of their differentiation is unknown and almost nothing is revealed about a possible application thereof to regenerative medicine. For example, engineering of which gene (and how differentiation occurs) can be applied to regenerative medicine remains unknown. Moreover, no one has succeeded in inducing differentiation into neurons by simply introducing a specific gene into immortalized cancer cells or ES cells for which it is very difficult to induce differentiation into neurons compared to neuronal stem cells. Among cancer cells, neuroblastoma cells (which are established from neuroblastoma, a kind of childhood cancer, developed from adrenal gland) are known to extend neuron-like projections when retinoic acid is added in the medium. However, neuroblastoma cells do not differentiate into true neurons having function to transmit electric signals. Known methods for differentiating cultured ES cells into neuronal stem cells and then to neurons include a method which adds retinoic acid into a medium (Fraichard A, et al.: J Cell Sci 108: 3181-8, 1995) and a method which adds basic fibroblast growth factor (bFGF) to cause differentiation to neurons at a relatively high rate (Okabe S, et al.: Mech Dev 59: 89-102, 1996). However, not all of the cells can differentiate into neurons by these methods and distinguishing between neuronal stem cells and neurons is required. Further, it is thought that these methods require 6 or more days to induce differentiation of ES cells into neurons.
In contrast to induction of differentiation into neurons, controlling differentiation is also an important technique. Regarding this technique, some cancer suppressor genes have been reported to have such a control function. Regarding control of neuronal differentiation using antisense of a gene which induces neuronal differentiation, we have reported that differentiation from neuronal stem cells into neurons is controlled by the antisense sequence (antisense) of VHL gene (Kanno H, et al.: Cancer Res 60: 2820-2824, 2000). Control of differentiation from cancer cells or ES cells has not been reported.
In the United States, clinical trials using fetal brain obtained by artificial termination of pregnancy for treating Parkinson's disease have been already conducted and a certain effect has been recognized. At the level of animal experiment, a trial, in which neuronal stem cells or ES cells are grafted to the brain or spinal cord, and the cells are allowed to differentiate into neurons, has been started to treat intractable neuronal diseases including not only Parkinson's disease, but also brain infarction, spinal cord injury and the like. Moreover, regeneration of peripheral nerve in vitro or in vivo in the form of bundles of nerve fibers has been attempted. However, since neurons do not principally divide and proliferate, it is difficult to form practical bundles of nerve. In nerve grafting to treat ruptured peripheral nerve, normally the autologous nerve of a lower limb is excised and grafted. However, production of artificial nerve for nerve grafting in place of autologous nerve has not been successful.